JP7745856B2 - Manufacturing method for biodegradable plastics using seaweed as a raw material - Google Patents

Description

NPMD NITE P-03085

The present invention relates to a method for producing polyhydroxyalkanoic acid (PHA) using seaweed as a raw material, which comprises step (a) of culturing a microorganism capable of alginate utilization and PHA synthesis in a medium containing seaweed.

Biodegradable plastics are polymeric materials that have the same properties and functions as ordinary plastics when not in contact with microorganisms, but when placed in the natural environment or a composting facility, they are assimilated by microorganisms and ultimately decompose into carbon dioxide and water. There are two types of biodegradable plastics: biodegradable petroleum-derived plastics, which use fossil resources as raw materials, and biodegradable bioplastics, which are made from biomass. With the increase in carbon dioxide emissions and global warming becoming problems, there is a demand for materials like bioplastics that are made from biomass, ultimately decompose into water and carbon dioxide, and do not accumulate in the environment.

The Japan Bioplastics Association states that a product must have a bioplastic content of 25% or more by mass in order to meet the biomass plastic identification standards. Products with a higher bioplastic content have a lower environmental impact. Examples of bioplastics made from 100% biomass raw materials include polylactic acid and polyhydroxyalkanoic acid (PHA).

PHAs are aliphatic polyesters synthesized by microorganisms. They serve physiological roles as carbon sources and energy compounds. They accumulate intracellularly when a carbon source is present under nutrient-depleted conditions, such as when nitrogen and phosphorus sources are scarce. One of the most studied PHAs, polyhydroxybutanoic acid (P(3HB)), was discovered in Bacillus megaterium by Dr. Lemoigne in 1925. Since then, its synthesis by over 300 species of bacteria, including hydrogen bacteria and cyanobacteria, has been reported. Due to its brittle nature (elongation at break of 5%), P(3HB) was considered inferior to polypropylene, which has a similar melting point and tensile strength. However, since Wallen and Rohwedder discovered a hydroxyalkanoic acid (HA) unit distinct from the 3-hydroxybutanoic acid unit (3HB) in 1974, various HAs have been reported, with over 150 types of monomer units currently reported. Furthermore, various studies have shown that the physical properties of PHA can be improved by incorporating monomer units other than 3HB to form a copolymer (Non-Patent Documents 1-6).

Previously, reports have been published on the microbial biosynthesis of PHAs using various organic substances as carbon sources. For example, Patent Document 1 discloses a method for producing polyhydroxyalkanoic acid by microorganisms, comprising culturing a microorganism belonging to the genus Cupriavidus, Ralstonia, or Alcaligenes that can assimilate lignin derivatives and/or their polyhydroxyalkanoic acid biosynthetic intermediates in a medium containing, as a carbon source, at least one substance selected from the group consisting of lignin derivatives and their polyhydroxyalkanoic acid biosynthetic intermediates, and recovering polyhydroxyalkanoic acid from the microbial cells. Patent Document 2 discloses a method for producing a polyhydroxyalkanoic acid copolymer, which comprises (i) culturing a transformant in which a polyhydroxyalkanoic acid polymerase mutant gene and a leucine metabolic gene derived from a Clostridium microorganism have been introduced into a microorganism in the presence of sugars, and (ii) collecting a polyhydroxyalkanoic acid copolymer containing 3-hydroxybutanoic acid and 3-hydroxy-4-methylvaleric acid as monomer units from the resulting culture, wherein the sugars are monosaccharides (glucose).

Patent Document 3 discloses a method for producing polyhydroxyalkanoic acid by microorganisms, obtained by polymerizing monomer units containing at least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, characterized in that the method uses fats and oils containing 41% by weight or more of lauric acid as a constituent fatty acid, and/or fatty acids as a carbon source, and cultures the microorganisms while adjusting the oxygen transfer rate so that the average hourly productivity of polyhydroxyalkanoic acid from the start of culture is 1.5 g/L/h or more. Other examples include a method for producing polyhydroxyalkanoic acid or polyhydroxyalkanoic acid copolymers using microorganisms that use sugars (glucose, fructose, sucrose), oils or fatty acids as the carbon source (Patent Documents 4 to 7); a method for fermentatively producing polyhydroxyalkanoic acid using microorganisms, in which the carbon source includes a carboxylic acid having four carbon atoms and/or an alcohol having four carbon atoms and derivatives thereof (Patent Document 8); a method for producing microbial polyesters, in which a microorganism capable of producing polyesters is cultured in a medium containing 1-hexene as the sole carbon source (Patent Document 9); and a method for producing polyhydroxyalkanoic acid, in which a microorganism capable of growing by assimilating acetic acid or a salt thereof and producing and accumulating polyhydroxyalkanoic acid is cultured in a medium containing acetic acid or a salt thereof as the sole carbon source (Patent Document 10).

On the other hand, PHA production using sugars and alcohol extracted from edible raw materials such as corn and sugarcane competes with food supplies, leading to rising prices. Therefore, research into utilizing unused biomass is expected to be a technology with low environmental impact. In recent years, seaweed has attracted global attention as one such unused biomass. In Japan, a large amount of seaweed is discarded during seaweed production and processing, with approximately 60% of the harvest in wakame seaweed processing being discarded (Non-Patent Document 7). This seaweed waste must be disposed of as industrial waste, which is a significant burden on fishermen and companies, and illegal dumping of seaweed waste is also a problem. Therefore, there are high hopes for using seaweed as a raw material for bioplastics with low environmental impact.

Japanese Patent Application Laid-Open No. 2015-077103 Japanese Patent Application Publication No. 2015-33 Japanese Patent Application Laid-Open No. 2013-9627 WO2012/102371 WO2009/147918 Japanese Patent Application Laid-Open No. 2009-225662 Patent Application No. 2006-238032 Japanese Patent Application Laid-Open No. 2009-225775 Japanese Patent Application Laid-Open No. 2001-178487 Japanese Patent Application Laid-Open No. 2001-178484

Kenichiro Matsumoto, Yoshiharu Doi, Biosynthesis of Biodegradable Polyesters: Current Status and Prospects, Journal of the Society of Synthetic Organic Chemistry, Vol. 61, No. 5, pp. 489-495, 2003 K. Sudesh, T Iwata, Sustainability of Biobased and Biodegradable Plastics, Clean 2008, 36(5-6), 433-442 K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, Prog. Polym. Sci., 25(10), 1503-1555, (2000) Tadahisa Iwata, Structure, Material and Biodegradability of Microbial Polyesters, Journal of the Crystallographic Society of Japan, 55, 188-196(2013) Wallen LL, Rohwedder WK, Poly-β-hydroxyalkanoate from activated sludge, Environ Sci Technol 8: 576-579 (1974) J. M. L. Dias, P. C. Lemos, L. S. Serafim, C. Oliveria, M. Eiroa, M. G. E. Albuquerque, A. M. Ramos, R. Oliveira, M. A M. Reis, Recent Advances in Polyhydroxyalkanoate Production by Mixed Aerobic Culture: From the Substrate to the Final Product, Macromol. Biosci., 6(11), 885-906 (2006) Shinichiro Fujii, Takashi Korenaga, Material flow analysis and zero-emission technology in the wakame seaweed processing industry, Journal of Environmental Science 13(5), 586-592, (2000) N. Azizi, G. Najafpour, H. Younesi, Acid pretreatment and enzymatic saccharification of brown seaweed for polyhydroxybutyrate (PHB) production using Cupriavidus necator, International Journal of Biological Macromolecules 101 (2017) 1029-1040

Previously, PHA biosynthesis using sugars, vegetable oils, alcohols, and other carbon sources has been reported, but because these carbon sources compete with food, there has been a need for the development of a method for producing bioplastics using unused biomass, such as seaweed waste. Prior art has reported the synthesis of PHA by a microorganism (Cupriavidus necator) using seaweed hydrolysate treated with acid and enzymes (Non-Patent Document 8). However, there is no information known about the production of bioplastics using seaweed, particularly brown algae, as a raw material. Therefore, the present invention aims to provide a method for producing PHA using seaweed, particularly brown algae, as a raw material, and to provide a novel microorganism capable of synthesizing PHA using seaweed as a raw material.

As a result of extensive research to solve the above problems, the inventors discovered a microorganism capable of synthesizing PHA using seaweed as a carbon source, and a method for producing PHA using this microorganism, thereby completing the present invention.
Specifically, the present invention provides the following [1] to [13].
[1] A method for producing polyhydroxyalkanoic acid (PHA) using seaweed as a raw material, comprising step (a) culturing a microorganism capable of assimilating alginic acid and synthesizing PHA in a medium containing seaweed.
[2] The method of [1], wherein the microorganism is a Cobetia bacterium.
[3] The method according to [1] or [2], wherein the seaweed is brown algae.
[4] The manufacturing method described in claim 3, wherein the brown algae are brown algae belonging to the Laminariales or Fucales orders.
[5] The production method according to any one of [1] to [4], wherein the culture step (a) is carried out in a medium having a pH in the range of 3.0 to 9.0 before the start of culture.
[6] The method according to any one of [1] to [5], wherein the microorganism is any one selected from the following 1) to 3):
1) The microorganism deposited under accession number NITE P-03085 .
2) Microorganisms deposited under accession number NITE P-02758 or NITE P-02759.
3) A microorganism having a 16S rRNA gene containing the following base sequence (a) or (b):
(a) A nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 3.
(b) A nucleotide sequence having 90% or more nucleotide sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 3.
[7] The method according to any one of [1] to [6], wherein the microorganism is an isolated microorganism, a deposited microorganism, or a genetically modified microorganism.
[8] The production method according to any one of [1] to [7], further comprising a step (b) of extracting PHA from the culture obtained in the culture step (a).
[9] The method according to any one of [1] to [8], wherein the PHA contains a 3-hydroxybutanoic acid monomer unit.
[10] The method according to [9], wherein the PHA is poly(3-hydroxybutanoic acid).
[11] A method for producing a bioplastic product, comprising the steps of: obtaining polyhydroxyalkanoic acid (PHA) by carrying out the method according to any one of [1] to [10]; and obtaining a plastic product containing the PHA obtained by the above step.
[12] Microorganism deposited under accession number NITE P-03085 .
[13] A bacterium of the genus Covetia having a 16S rRNA gene containing the following nucleotide sequence (a) or (b):
(a) The base sequence set forth in SEQ ID NO: 1.
(b) A nucleotide sequence having 90% or more nucleotide sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1.

The present invention provides a method for producing bioplastics using seaweed as a carbon source.

FIG. 1 shows the results obtained by GC-MS for the 5-25-4-2 strain. FIG. 2 shows the results of ¹ H-NMR analysis of the 5-25-4-2 strain. FIG. 3A shows the nucleotide sequence of the 16S rRNA gene of the 5-25-4-2 strain (SEQ ID NO: 1). FIG. 3B shows the nucleotide sequence of the 16S rRNA gene of the 5-11-6-3 strain (SEQ ID NO: 2). FIG. 3C shows the nucleotide sequence of the 16S rRNA gene of the 5-28-6-1 strain (SEQ ID NO: 3). Figure 4 shows the results of ¹ H-NMR analysis of the polymer obtained from strain 5-11-6-3 using wakame seaweed as a carbon source. FIG. 5 shows the results of ¹ H-NMR analysis of the polymer obtained from strain 5-25-4-2 using kelp as a carbon source. FIG. 6 shows the biosynthetic pathway of PHA.

The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. Note that in this specification, numerical ranges expressed using "to" refer to ranges that include the numerical values before and after "to" as the lower and upper limits.

<PHA manufacturing method>
The method of the present invention for producing polyhydroxyalkanoic acid (PHA) using seaweed as a raw material comprises the step (a) of culturing a microorganism capable of alginate assimilation and PHA synthesis in a medium containing seaweed.
By carrying out the production method of the present invention, it is possible to produce PHA, a biodegradable plastic, using seaweed as a carbon source. According to the present invention, PHA can be produced using seaweed as a raw material, making it possible to produce bioplastic products that have a low impact on the global environment.

(Culture medium containing seaweed)
In the production method of the present invention, PHA can be produced by culturing microorganisms capable of alginate utilization and PHA synthesis in a medium containing seaweed. Because seaweed, particularly brown algae, contains alginic acid, it is believed that the microorganisms synthesize PHA from alginic acid. Alginic acid is a polysaccharide found in brown algae and other organisms, and is a linear polymer of two types of uronic acid: β-D-mannuronic acid (M) and its C-5 epimer, α-L-guluronic acid (G).

The inventors have previously discovered that PHA can be produced from alginic acid using microorganisms capable of alginic acid assimilation and PHA synthesis. In the present application, the inventors have discovered that PHA can be produced using seaweed as a raw material using microorganisms capable of alginic acid assimilation and PHA synthesis. In other words, because PHA can be produced using seaweed itself as a raw material, the step of extracting alginic acid from seaweed can be omitted. Furthermore, because seaweed is an abundant biomass in Japan, which is poor in energy resources, it may become unnecessary to rely on imports for the supply of PHA raw materials in the future.

In seaweed, alginic acid (AL-) is trapped as an inorganic ion salt with calcium ions, etc. According to the disclosure of JP 2017-210534 A, for example, alginic acid can be extracted from seaweed by adding sodium carbonate to seaweed, heating the seaweed, and exchanging sodium ions (Na ⁺ ) for calcium ions (Ca ²⁺ ) to extract alginic acid as water-soluble sodium alginate, then removing calcium carbonate from the alginic acid extract, and either acidifying the alginic acid extract from which calcium carbonate has been removed to precipitate alginic acid, or adding calcium ions to precipitate calcium alginate, which is then filtered to obtain alginic acid or calcium alginate.
According to the present invention, PHA can be produced using seaweed as a raw material, so that extraction of alginic acid from seaweed does not need to be included as a pre-step, and PHA can be produced by a simple method.

In addition, PHA synthesis using a microorganism (Cupriavidus necator) using a seaweed hydrolysate hydrolyzed with acid and enzymes as a raw material has been reported (Non-Patent Document 8). Specifically, Non-Patent Document 8 uses a hydrolysate obtained by heating seaweed from the genus Sargassum (Sargassum sp.) in the presence _of 0.15% _NH2SO4 at 121°C for 30 minutes and further treating it with cellulase and cellobiase. This hydrolysate contains reducing sugars, particularly glucose that is thought to be derived from cellulose. On the other hand, the PHA production method of the present invention allows seaweed to be used as a raw material without pretreatment such as hydrolysis, thereby enabling PHA to be produced in a simpler manner.

In the production method of the present invention, the seaweed content of the culture medium can be set appropriately depending on the type of seaweed and the type of microorganism, but is preferably in the range of 0.5% to 20% by mass, more preferably 1% to 10% by mass, and particularly preferably 1% to 5% by mass, on a dry weight basis.

In one embodiment of the present invention, before the start of cultivation, the pH of the seaweed-containing medium can be adjusted to a range of about 3.0 to 9.0, about 3.0 to 8.0, about 3.0 to 7.0, about 3.0 to 6.5, about 4.0 to 6.5, about 4.5 to 6.5, or about 4.0 to 6.5.

Seaweed that can be used in the PHA production method of the present invention is a macroalgae selected from brown algae, red algae, green algae, etc., with brown algae being preferred. This is because brown algae are rich in alginic acid. Examples of brown algae include brown algae belonging to the orders Laminariales, Fucales, Aldehydes, Azomales, Chordatales, Sclerotiales, Foeniculales, Pectinales, Dictyotales, Saffronales, Pyrrhizales, and Parsnipales, but are not limited to these orders. Brown algae that grow in the oceans of various parts of the world can be used. Examples of brown algae belonging to the order Laminariales include the genera Laminaria, Kjellmaniella, Ecklonia, Eisenia, Eckloniopsis, Agarum, Costaria, Arthrothamnus, Macrocystis, and Hedophyllum from the family Laminariaceae, the genera Undaria and Alaria from the family Alariaceae, the genus Chorda from the family Chordaceae, and the genus Lessonia from the family Lessoniaceae. Examples of brown algae in the Fucales order include the genera Sargassum (including S. fusiforme), Coccophora, Cystoseira, Myagropsis, and Turbinaria in the Sargassaceae family, and the genera Fucus and Pelvetia in the Fucaceae family.

Brown algae belonging to the order Estocarpales include the genera Ectocarpus, Botrytella, Giffordia, and Pilayella of the family Estocarpaceae. Brown algae belonging to the order Ralfsiales include the genus Ralfsia of the family Ralfsiaceae. Brown algae in the order Chordariales include the genera Papenfusiella, Tinocladia, Acrothrix, Analipus, Chordaria, Cladosiphon, Eudesme, Myelophycus, Saundersella, and others in the family Chordariaceae. Examples include the genus Sphaerotrichia, the genera Elachista and Halothrix from the family Elachistaceae, the genera Leathesia and Petrospongium from the family Leathesiaceae, the genus Nemacystis from the family Nemacystaceae, and the genus Ishige from the family Ishigeaceae. Examples of brown algae belonging to the Scytosiphonales order include the genera Colpomenia, Petalonia, Scytosiphon, Akkesiphycus, Coilodesme, Endarachne, Hydroclathrus, and Melanosiphon from the Scytosiphonaceae family, the genus Chnoospora from the Chnoosporaceae family, and the genus Punctaria from the Punctariaceae family.

Brown algae belonging to the order Dictoysiphonales include the genus Dictyosiphon in the family Dictyosiphonaceae. Brown algae belonging to the order Cutleriales include the genus Cutleria in the family Cutleriaceae. Brown algae belonging to the order Sphacelariales include the genus Sphacelaria in the family Sphacelariaceae. Examples of brown algae belonging to the Dictyotales order include the genera Dictyopteris, Dictyota, Dilophus, Pachydictyon, Padina, Spatoglossum, Distromium, Homoeostrichus, Pockockiella, Stypopodium, and Zonaria from the Dictyotaceae family. Examples of brown algae belonging to the Sporochnales order include the genera Carpomitra and Sporochnus from the Sporochnaceae family. Examples of brown algae belonging to the Desmarestiales order include the genus Desmarestia in the family Desmarestiaceae. Examples include Syringoderma in the family Syringodermataceae in the order Syringodermatales.

In one embodiment of the present invention, seaweed that can be used in the PHA production method include brown algae belonging to the Laminariales order, such as kelp (Laminaria sp.), wakame (Undaria sp.), and Macrocystis sp., and brown algae belonging to the Fucales order, such as Sargassum sp. Brown algae such as kelp and wakame have been reported to contain 20-40% alginic acid per dry weight (Hiroyuki Takeda et al., Energy Environ. Sci., 4, 2575-2581, (2011)). Furthermore, the alginic acid content in brown algae remains nearly constant throughout the year (T. Kimura et al., Nippon Suisan Gakkaishi, 73(4), 739-744, (2007)). Therefore, brown algae are considered a highly promising biomass source.

Seaweed may be used fresh or dried (freeze-dried, sun-dried, naturally dried, blown-air dried, dehumidified air dried, hot-air dried, far-infrared dried, etc.). It may also be boiled, steamed, or otherwise heat-treated. Seaweed that has been desalted may be used to maintain the medium at a salt concentration and pH suitable for the growth of microorganisms, such as the Covetia bacteria described below. Seaweed may be divided or cut into appropriate sizes, or pulverized using a mortar and pestle, mixer, grinder, or the like, and then mixed with the medium. In one embodiment of the present invention, seaweed can be pulverized using a mortar and pestle, mixer, grinder, or the like, and then added to and mixed with the medium. This is believed to improve the efficiency of PHA synthesis. Seaweed may be shredded or crushed to sizes of approximately 1 to 3 square centimeters or 2 to 3 square millimeters, or to sizes of 0.5 square millimeters or less.

The seaweed-containing medium is a seaweed-containing medium for PHA synthesis. Any medium capable of growing microorganisms capable of alginate assimilation and PHA synthesis can be used. For example, in addition to the seaweed described above, it can contain an inorganic nitrogen source (e.g., ammonium sulfate, ammonium nitrate, urea). The medium can also contain minerals and trace elements, such as salts of phosphorus (P), sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), cobalt (Co), copper (Cu), zinc (Zn), and nickel (Ni). Specific examples of substances containing minerals and trace elements include calcium chloride, magnesium sulfate, potassium nitrate, sodium chloride, iron (III) chloride, calcium chloride, cobalt chloride, copper (II) sulfate, nickel (II) chloride, and chromium (III) chloride. In a preferred embodiment of the present invention, the seaweed-containing medium for PHA synthesis is a nitrogen-limited medium or a nitrogen-limited inorganic salt medium containing seaweed.

(microorganisms)
The method for producing PHA of the present invention can be carried out using microorganisms capable of alginate assimilation and PHA synthesis. Alginate assimilation is the ability to grow using alginate as a sole carbon source. PHA synthesis is the ability to biosynthesize PHA. Microorganisms capable of alginate assimilation and PHA synthesis can be said to be microorganisms capable of synthesizing PHA using alginate as a sole carbon source. Microorganisms capable of alginate assimilation and PHA synthesis may be microorganisms that can assimilate alginate decomposition products in addition to alginate and synthesize PHA.

Whether a microorganism has the ability to utilize alginate can be determined by culturing it in a medium containing alginate as the sole carbon source and determining whether the microorganism grows. For example, a test microorganism can be cultured in a medium containing 1% sodium alginate by mass as the sole carbon source under conditions suitable for the growth of the test microorganism (temperature, pH, oxygen, sodium salt concentration, etc.). If the test microorganism grows, it can be determined that the test microorganism has the ability to utilize alginate. Growth of the test microorganism can be measured by the turbidity method, plate culture method, dry cell weight method, etc.

The ability of a microorganism to synthesize PHA can be determined by culturing it in a nitrogen- or phosphorus-limited medium and determining whether it synthesizes PHA. For example, a test microorganism can be cultured in a nitrogen-limited inorganic salt medium containing 1-2% glucose as the sole carbon source under conditions suitable for its growth. If the test microorganism accumulates PHA within its body, the test microorganism can be determined to have the ability to synthesize PHA. PHA accumulation can be confirmed by Nile Red or Nile Blue A staining, as well as by observing intracellular PHA granules with a transmission electron microscope, ^1H -NMR analysis of chloroform extracts obtained from dried cells, and gas chromatography or gas chromatography-mass spectrometry (GC-MS) of ethanolyzed chloroform extracts. Furthermore, if the accumulated PHA is PHB, it can be converted to crotonic acid with sulfuric acid and then analyzed by high-performance liquid chromatography to determine PHB accumulation.

The microorganisms capable of alginate utilization and PHA synthesis may be, for example, marine bacteria belonging to the Proteobacteria phylum, or bacteria belonging to the Halomonadaceae family. Bacteria belonging to the Proteobacteria phylum are Gram-negative bacteria. Marine bacteria are bacteria that live in the ocean and can grow well in seawater.

The microorganisms capable of alginate utilization and PHA synthesis may be bacteria belonging to the genus Cobetia. Examples of bacteria belonging to the genus Cobetia include Cobetia marina, Cobetia pacifica, Cobetia amphilecti, and Cobetia litoralis, as well as undescribed species inferred to belong to the genus Cobetia based on 16S rDNA sequences. In the present invention, the microorganism capable of alginate utilization and PHA synthesis can be selected from the genus Cobetia, preferably Cobetia marina and its related species (including undescribed species), more preferably from the species to which the 5-25-4-2, 5-11-6-3, and 5-28-6-1 strains described in the Examples below belong, and it is particularly preferred to use the 5-25-4-2, 5-11-6-3, and 5-28-6-1 strains. Kobetia marina was previously classified as Arthrobacter marinus, Pseudomonas marina, Dalya marina, and Halomonas marina (Arahal DR, et al., (December 2002) Systematic and Applied Microbiology. 25 (2): 207-11).

In one embodiment of the present invention, the microorganism having the ability to utilize alginic acid and synthesize PHA may be any one selected from the following 1) to 3).
1) The microorganism deposited under accession number NITE P-03085 ( accession number NITE AP-03085 ) .
2) Microorganisms deposited under accession number NITE P-02758 or NITE P-02759.
3) A microorganism having a 16S rRNA gene containing the following base sequence (a) or (b):
(a) A nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 3.
(b) A nucleotide sequence having 90% or more nucleotide sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 3.

The above-mentioned microorganism can be a microorganism that has a 16S rRNA gene containing a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 3, and that has the ability to utilize alginate and synthesize PHA. Furthermore, the above-mentioned microorganism can be a microorganism belonging to the genus Covetia that has a 16S rRNA gene containing a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 3, and that has the ability to utilize alginate and synthesize PHA. The microorganism may also be a microorganism belonging to the genus Covetia, which has a 16S rRNA gene containing a base sequence in which one or several bases have been substituted, inserted, deleted, and/or added in the base sequence set forth in any one of SEQ ID NOs: 1 to 3, and which has the ability to utilize alginate and synthesize PHA.

The base sequence can be analyzed using standard methods. For example, methods for amplifying the 16S rRNA gene from microbial DNA include, but are not limited to, PCR using primers. The amplified product obtained by PCR can be subjected to DNA sequence analysis using a DNA sequencer or the like.

Sequence identity is defined as the percentage of bases that are identical between two sequences after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Sequence identity can be determined using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ClustalX, or Megalign (DNASTAR) software.

As used herein, the range of "one or several" in the "base sequence in which one or several bases have been substituted, inserted, deleted and/or added" is not particularly limited, but means, for example, about 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3.

The microorganism may be an isolated microorganism, a deposited microorganism, or a genetically modified microorganism. The Covetia bacterium may also be an isolated strain, a deposited strain, or a genetically modified strain. An isolated microorganism or strain is a microorganism or strain isolated from a natural sample. Examples of natural samples include seawater, marine organisms, seaweed, brackish water, brackish water organisms, rocks, soil, and sand. A natural sample can be cultured in any medium, such as ZoBell 2216E seawater medium, MS medium, or Daigo IMK medium, at 10°C to 42°C for 24 to 72 hours at a pH of 5 to 10, and colonies can be isolated. Prior to culturing in ZoBell 2216E seawater medium or MS medium, the bacteria can be precultured in filtered seawater. The isolated colonies are cultured in an alginate-containing medium, and colonies that show growth in the alginate-containing medium and are determined to be synthesizing PHA can be selected. Nile Red is a hydrophobic dye that reacts with hydrophobic substances such as neutral lipids and PHA in living organisms, turning red in color, and can therefore be used to screen for microorganisms capable of synthesizing PHA.

Furthermore, for PHA synthesis, PHA can be extracted from dried microbial cells with an organic solvent such as chloroform, and then precipitated by adding an organic solvent such as methanol or hexane. Screening can then be performed using analytical techniques such as gas chromatography, liquid chromatography, NMR, and IR. Furthermore, in some cases, the strains of isolated colonies can be identified based on their 16S rDNA sequence or bacteriological properties.

The deposited microorganisms or strains can be obtained by selecting microorganisms capable of alginate utilization and PHA synthesis using the same techniques as those described for isolated microorganisms. For example, microorganisms capable of alginate utilization and PHA synthesis can be selected from deposited marine bacteria belonging to the family Halomonadaceae. Furthermore, microorganisms capable of alginate utilization and PHA synthesis can be selected from deposited microorganisms belonging to the genus Covetia.
The genetically modified microorganism or strain may be, for example, a microorganism or strain in which the alginate decomposing enzyme gene, PHA synthase gene and/or genes related thereto have been modified.

(PHA)
The polyhydroxyalkanoic acid (PHA) for which the production method is provided by the present invention is a polyester known to accumulate in the bodies of certain microorganisms, and has the following chemical formula:
[In the formula, R may be the same or different and is a linear or branched alkyl group having 1 to 14 carbon atoms, and n is the number of repetitions and is an integer of 2 or more, preferably an integer of 100 or more, and preferably an integer of 100,000 or less].
Specific examples of PHAs include those having the following chemical formula:
Examples of the polyhydroxybutanoic acid include polyhydroxybutanoic acid (P(3HB) or PHB) having the formula: Representative copolymers include poly(3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)], poly(3-hydroxycaproic acid-co-3-hydroxycaproic acid) [P(3HB-co-3HHx)], poly(3-hydroxypropionic acid) [P(3HB-co-3HP)], poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) [P(3HB-co-4HB)], poly(3-hydroxybutanoic acid-co-lactic acid) [P(3HB-co-LA)], and poly(3-hydroxybutanoic acid-co-glycolic acid) [P(3HB-co-GL)].

The biosynthetic pathway of P(3HB), a type of PHA, has been extensively studied in Ralstonia eutropha. It has been reported that two molecules of acetyl-CoA are condensed to acetoacetyl-CoA by β-ketothiolase (PhaA), which is then reduced to (R)-3-hydroxybutyryl-CoA by NADPH-dependent acetoacetyl-CoA reductase (PhaB), followed by polymerization by PHA synthase (PhaC) (Figure 6) (Yamada, M. et al., Oleoscience, 5 (11), 523-532 (2005); K. Sudesh et al., Prog. Polym. Sci. 25 (2000) 1503-1555). Furthermore, the biosynthesis of medium-chain 3HA (6-14 carbon atoms) has been studied in Pseudomonas bacteria and Aeromonas caviae, and it has been reported that the fatty acid beta-oxidation and biosynthetic pathway are involved (Figure 6) (Hiromi Matsuzaki et al., Journal of the Japan Oil Chemists' Society, 48(12), 1353-1363, 1999; T. Fukui et al., J. Bacteriol, 180(3), 667-673, 1998; T. Fukui et al., J. Bacteriol, 179(15), 4821-4830, 1997).

The PHA produced by the method of the present invention can have a molecular weight of 200,000 to 2,000,000. The PHA produced by the method of the present invention can contain a 3-hydroxybutanoic acid monomer unit. Examples of PHAs containing a 3-hydroxybutanoic acid monomer unit include poly(3-hydroxybutanoic acid-co-3-hydroxypropionic acid) [P(3HB-co-3HP)], poly(3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)], poly(3-hydroxybutanoic acid-co-3-hydroxycaproic acid) [P(3HB-co-3HHx)], poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) [P(3HB-co-4HB)], poly(3-hydroxybutanoic acid-co-lactic acid) [P(3HB-co-LA)], and poly(3-hydroxybutanoic acid-co-glycolic acid) [P(3HB-co-GL)].
The PHA for which the production method is provided by the present invention can be poly(3-hydroxybutanoic acid).

(Culture conditions)
In the production method of the present invention, cultivation for PHA biosynthesis can be carried out at a temperature in the range of about 20°C to 40°C and a pH in the range of about 3 to 9. The cultivation temperature and pH can be appropriately adjusted depending on the microorganism being cultivated. For example, among the above-mentioned microorganisms, the Covetia bacteria deposited under Accession No. NITE P-03085 ( Accession No. NITE AP-03085 ) , Accession No. NITE P-02758, and NITE P-02759 are microorganisms isolated by screening under acidic conditions, and therefore, cultivation for PHA synthesis using these bacteria can be carried out in an acidic medium.

When marine bacteria are used as microorganisms capable of alginate utilization and PHA synthesis, the concentration of sodium chloride in the culture medium can be set appropriately depending on the type of marine bacteria, etc., but it is preferably adjusted to a range of 0.5% to 20% by mass, more preferably 1% to 10% by mass, and particularly preferably 1% to 8% by mass.

In the production method of the present invention, cultivation for PHA synthesis is carried out under aerobic conditions, and an aeration-agitation type cultivation tank or a bubble column type cultivation tank can be used. The size of the cultivation tank can be, for example, 150 milliliters or more. Furthermore, when culturing on an industrial scale, the size of the cultivation tank can be, for example, 30 liters or more, 50 liters or more, or 100 liters or more. Cultivation can be carried out while continuously or intermittently supplying a carbon source and other nutrient sources. The carbon source concentration in the medium can be set in the range of about 0.5% to about 5.0% by mass. The agitation speed can be, for example, 100 to 120 rpm. The cultivation period can be set in the range of 1 to 7 days, but is not limited to this. Cultivation can be carried out while controlling conditions such as temperature, pH, seaweed content, aeration volume, agitation speed, and cultivation time.

The fungal cells to be inoculated into the medium may be previously seed cultured or pre-cultured. Alginic acid may be added to the medium used for seed culture or pre-culture. Other ingredients may include inorganic nitrogen sources (e.g., ammonium sulfate, ammonium nitrate, urea), minerals, and trace elements, such as salts of phosphorus (P), sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), cobalt (Co), copper (Cu), zinc (Zn), and nickel (Ni). Specific examples of substances containing minerals and trace elements include calcium chloride, magnesium sulfate, potassium nitrate, sodium chloride, iron (III) chloride, calcium chloride, cobalt chloride, copper (II) sulfate, nickel (II) chloride, and chromium (III) chloride.

(PHA recovery)
PHA accumulated within microbial cells can be recovered by known methods (see, for example, Patent Document 3). For example, bacterial cells are separated from the culture using a separation means such as a centrifuge, washed with distilled water, methanol, or the like, and dried. PHA is extracted from the dried bacterial cells using an organic solvent such as chloroform. Bacterial components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. The supernatant is then removed by filtration or centrifugation, and the PHA can be recovered by drying. In the present invention, since the medium contains seaweed, any filtration means may be used to remove the seaweed from the medium before centrifuging the bacterial cells.

The present invention provides a method for producing bioplastic products, which includes the steps of obtaining polyhydroxyalkanoic acid (PHA) by carrying out the above-mentioned production method, and obtaining a plastic product containing the PHA obtained in the previous step. PHA is a biodegradable plastic, and using it to produce bioplastic products is expected to solve the problem of microplastics remaining in the environment, which has recently become a cause of marine pollution.

In one embodiment of the present invention, plastic products containing 25% by weight or more of PHA can be produced. The Japan Bioplastics Association states that a product must have a bioplastic content of 25% by weight or more in order to comply with the Biomass Plastic Identification Standards. The bioplastic content is the ratio of the mass of biomass plastic components in a biomass plastic product to the total mass.

The PHA produced by the manufacturing method of the present invention can be used as a raw material for various plastic products. Examples of plastic products containing PHA include films and sheets that can be used as packaging for frozen foods and cushioning materials, stationery such as ballpoint pens, disposable tableware such as straws, forks, spoons, and plates, civil engineering and construction materials, multipurpose films for agricultural and fishery use, seedling pots, fishing lines, and fishing nets.

(Covetia bacteria)
The present inventors isolated the microbial strains 5-25-4-2, 5-11-6-3, and 5-28-6-1 from natural samples collected from Goishi Beach in Ofunato Bay, Ofunato City, Iwate Prefecture. The microbial strains 5-25-4-2, 5-11-6-3, and 5-28-6-1 have 16S rRNA genes containing the nucleotide sequences set forth in SEQ ID NOS: 1, 2, and 3, respectively. Analysis of a sequence database using the homology search program BLAST (Altschul SF et al., 1990 Basic local alignment search tool. J. Mol. Biol. Vol. 215, pp. 403-410) based on the nucleotide sequences of the 16S rRNA genes suggests that the three strains may belong to the genus Covetia.

Covetia bacteria are reported to be aerobic and grow at temperatures between 4 and 42°C and pH between 4 and 11 (Romanenko LA et al., International Journal of Systematic and Evolutionary Microbiology, 63, 288-297, (2013)). They have also been reported to accumulate P(3HB) intracellularly using glucose as a carbon source (Romanenko LA et al. 2013; Arahal DR et al., Appl. Microbiol. 25, 207-211, 2002). However, prior to this application, there had been no reports of PHA accumulation using seaweed as a carbon source.

A comparison of the phenotype characteristics of strain 5-25-4-2 with strain DSM4741 (Arahal DR et al., 2002, supra), the type strain of Cobetia marina, revealed that Cobetia marina can grow in a medium with a 0% NaCl concentration, while strain 5-25-4-2 cannot grow in a medium with a 0% NaCl concentration. Furthermore, the strains differ in their ability to assimilate L-arabinose and D-mannose (Table 21 in Example 2). Therefore, strain 5-25-4-2 is a Cobetia bacterium, and although it is closely related to Cobetia marina, it may be a different species.

The present invention provides a bacterium of the genus Covetia that has the ability to utilize alginate and synthesize PHA.
The present invention provides a bacterium of the genus Covetia having a 16S rRNA gene comprising the following base sequence (a) or (b):
(a) the nucleotide sequence set forth in SEQ ID NO: 1;
(b) A nucleotide sequence having 90% or more nucleotide sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1.

The present invention provides a bacterium of the genus Covetia having a 16S rRNA gene containing the following nucleotide sequence (a) or (b), and having the ability to utilize alginate and synthesize PHA:
(a) the nucleotide sequence set forth in SEQ ID NO: 1;
(b) A nucleotide sequence having 90% or more nucleotide sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1.

The nucleotide sequence and nucleotide sequence identity can be analyzed by the methods described above, and whether a bacterium has the ability to utilize alginate and/or synthesize PHA can be determined by the methods described above.
The Covetia bacteria of the present invention have the ability to utilize alginic acid and synthesize PHA, and therefore can be used in a method for producing bioplastics using seaweed as a raw material.

(P(3HB) accumulation rate)
When Covetia bacteria are cultured in a medium containing seaweed, they (e.g., strains 5-25-4-2, 5-11-6-3, and 5-28-6-1) can efficiently synthesize and accumulate P(3HB). The P(3HB) accumulation rate can be calculated, for example, by gas chromatography-mass spectrometry (GC-MS) using a calibration curve with 3HB ethyl as a standard substance, followed by dividing the P(3HB) amount by the dry cell weight. In one embodiment of the present invention, by culturing Covetia bacteria in a medium containing seaweed, Covetia bacteria (e.g., strains 5-25-4-2, 5-11-6-3, and 5-28-6-1) can synthesize and accumulate P(3HB) at P(3HB) accumulation rates of 5% by mass, 10% by mass, 15% by mass, 20% by mass, 25% by mass, 30% by mass, 35% by mass, 40% by mass, 45% by mass, or 50% by mass or more.

In the examples below, it was shown that when Covetia strain 5-11-6-3 bacteria were cultured for 24 hours in a medium containing 5% by mass of dried kelp, the P(3HB) accumulation rate was 48%. When Covetia strain 5-11-6-3 bacteria were cultured for 24 hours in a medium containing 3% sodium alginate, the P(3HB) accumulation rate was approximately 46% (Patent Application No. 2019-142249). This demonstrates that Covetia bacteria can synthesize and accumulate P(3HB) with efficiency equal to or greater than that achieved when using seaweed as a raw material, as compared to when using sodium alginate.

(Microorganism deposit)
The 5-25-4-2 strain was deposited with the depository institution by Iwate University (address: 3-18-8 Ueda, Morioka, Iwate Prefecture) as follows: The 16S rRNA gene of the 5-25-4-2 strain has the base sequence set forth in SEQ ID NO: 1.
(i) Recipient: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (NITE-NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan)
(ii) Received on November 29, 2019
(iii) Accession number NITE P-03085 ( Receipt number NITE AP-03085 ) (Cobetia sp. IU190790JP01(5-25-4-2) strain)

The 5-11-6-3 strain has been deposited by Iwate University (Address: 3-18-8 Ueda, Morioka City, Iwate Prefecture) with the following depository institution: The 16S rRNA gene of the 5-11-6-3 strain has the base sequence set forth in SEQ ID NO:2.
(i) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (NITE-NPMD)
(Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan)
(ii) Trust date (deposit date): August 8, 2018
(iii) Accession number NITE P-02758 (Cobetia sp. IU180733JP01 (5-11-6-3) strain)

The 5-28-6-1 strain has been deposited with the following depository by Iwate University (Address: 3-18-8 Ueda, Morioka City, Iwate Prefecture, Japan). The 16S rRNA gene of the 5-28-6-1 strain has the base sequence set forth in SEQ ID NO:3.
(i) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (NITE-NPMD)
(Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan)
(ii) Trust date (deposit date): August 8, 2018
(iii) Accession number NITE P-02759 (Cobetia sp. IU180733JP01 (5-28-6-1) strain)

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%" is based on mass, and numerical ranges are stated as including their endpoints.

Reagents and Apparatus The reagents and apparatus used in the examples are as follows.

Example 1
<Isolation of Covetia bacteria>
Covetia bacteria were isolated and identified from natural samples using the following procedure.
The natural samples consisted of seawater, seaweed, shells, and sea slugs collected from the Goishi Coast area of Ofunato City, Iwate Prefecture on June 20, 2017. The collected natural samples were cultured for 2-3 days (scale: 5 mL test tube, temperature: 30°C, stirring speed: 111 rpm, medium: ZoBell 2216E seawater medium, pH: 5).

To obtain single colonies from seawater culture medium containing natural samples, single bacterial isolation was performed using the medium shown in Table 7 at a culture temperature of 30°C for three days. Multiple colonies with different colors, morphologies, and sizes were obtained.

Next, as a primary screening step, strains obtained by single cell isolation were cultured in a medium containing Nile Red to select candidate strains for PHA synthesis. Nile Red is a hydrophobic dye that reacts with neutral lipids within cells to produce a red color, making it possible to visually identify candidate strains. Two types of medium, one containing Nile Red and one not, were prepared, and the color change was observed with the naked eye by inoculating bacteria onto both plates. Red-colored colonies were stored at 4°C until the next experiment.

The composition of the Nile Red-containing medium is shown in Table 8. The medium was autoclaved (121°C, 15 min). Trace Element was filter-sterilized (Minisart®-RC25, sartorius, RC, pore size 0.45 μm). Sodium alginate was autoclaved (121°C, 15 min) separately from the medium. Nile Red was dissolved in dimethyl sulfoxide and used without sterilization. The final concentration of sodium alginate in the medium was 1%. Colonies with different colors and morphologies were selected from ZoBell 2216E seawater plates, picked with a toothpick, and inoculated onto two plates. The plates were incubated at 30°C for 5 days, and the Nile Red-containing plates were incubated in the dark. The plates were visually observed daily, and multiple red-colored strains were identified.

Next, a secondary screening was performed using GC-MS. After culturing the strains selected in the primary screening, PHA polymers were extracted, and the extracts were ethanolyzed. PHA monomers were detected by GC-MS for further selection.

(Culturing) PHA-synthesizing candidate strains obtained in the primary screening were cultured in test tubes (10 mL). The medium listed in Table 9 was autoclaved (121°C, 15 min). Trace Element and sodium alginate were sterilized separately using the same method as above and then added to the medium. Colonies of the PHA-synthesizing candidate strains obtained in the primary screening were isolated with toothpicks and placed in the medium, along with the toothpicks, for shaking culture. Sodium alginate was used as the carbon source, with a final concentration of 1%. Culture was performed at 30°C with an agitation speed of 111 rpm for 72 hours, with the culture time extended for samples with slow growth.

(PHA extraction) After incubation, the pH and OD ₆₆₀ of the culture medium were measured, and the cells were harvested by suspending twice in RO water (water treated with a reverse osmosis membrane) and centrifuging three times. Centrifugation was performed at 8,490 × g or 7,670 × g. The harvested cells were pre-frozen at -30°C. The tube containing the cells was then capped with parafilm, punctured, and freeze-dried for at least 24 hours in a freeze dryer. After freeze-drying, the dried cells were weighed and crushed. After crushing, the cells were transferred to a screw-cap test tube, and 5 mL of chloroform was added per 200–300 mg of cells. After vortexing, the mixture was heated at 70°C for two days. Vortexing was performed as needed. After heating, the cells were removed by passing through a filter, and the filtrate was heated to dryness in a fume hood. The weight of the extract was determined by weighing the screw-cap test tube before and after filtration. The dried sample was stored at room temperature.

(GC-MS Sample Preparation) 1 mL of chloroform was added to the extract and dissolved by heating. 3.4 mL of 99.5% ethanol and 400 μL of HCl (stock solution) were added, vortexed for 1 minute, and heated in a heat block at 100°C for 4 hours (ethanolysis treatment) (Y. Arai, et al., Plant Cell Physiol. 43(5), 555-562, (2002)). During heating, vortexing was performed for 1 minute every 30 minutes. After cooling on ice, 4 mL of a 0.65 M NaOH + 0.9 M NaCl mixture and 2 _mL of 0.25 M _Na2HPO4 were added, vortexed for 1 minute, and the chloroform layer was confirmed to be neutral using pH paper. The solution was then centrifuged at 800 × g for 5 minutes, and the chloroform layer was collected at the bottom. The chloroform layer was passed through a column made by packing glass wool and _NaSO in a Pasteur pipette, _and then dehydrated by transferring it to a screw-cap test tube containing molecular sieves that had been preheated at 180°C for 2 hours. This was used as a sample and was stored at -30°C until analysis.

To measure the amount of synthesized PHA, P(3HB), one of the most common PHAs, was subjected to ethanolysis, and a calibration curve was created using 3HB ethyl as a standard substance. To create the calibration curve, P(3HB) was diluted with chloroform to prepare concentrations of 10, 50, 100, 500, and 1000 μg/mL, and each ethanolyzed sample was used.

(GC-MS method conditions)
An HP-5 column (length 30 m, film thickness 0.25 μm, inner diameter 0.25 mm) was used. The analytical conditions are as shown in Table 10.

A peak in the mass spectrum of the 3HB ethyl standard substance was confirmed at a retention time of approximately 4.7. A peak in the mass spectrum of 3HB ethyl, thought to be derived from P(3HB), was detected in multiple samples. Samples that showed peaks between retention times of 4.76 and 5.00 were selected as candidate strains for PHA synthesis. 3HB ethyl, an ethanolysis product of P(3HB), one of the most common PHAs, was detected in multiple strains. Of the multiple strains that showed peaks between retention times of 4.76 and 5.00, the analysis results for strain 5-25-4-2 are shown in Figure 1.

The amount of P(3HB) was determined from a calibration curve using 3HB ethyl as a standard substance, and the P(3HB) accumulation rate was calculated by dividing the amount of P(3HB) by the dry cell weight (Table 11).

Next, ^1H -Nuclear Magnetic Resonance ( ^1H -NMR) analysis was performed to confirm polymer synthesis. The 5-25-4-2 strain was cultured in a Sakaguchi flask for scale-up, and a chloroform extract was obtained from the culture. This was dissolved in a small amount of chloroform, and an excess (more than 10 times) of hexane was added to precipitate the polymer, which was then purified. 2 mL of 1 vol% TMS-containing deuterated chloroform (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the purified polymer, and the polymer was dissolved by heating.
^1H -NMR analysis revealed peaks specific to P(3HB) homopolymer (1.26-1.28, 2.44-2.64, 5.24-5.28 ppm), but no peaks for the monomer 3HB were detected. Figure 2 shows the analysis results for strain 5-25-4-2. The 3HB peaks were observed at 1.20-1.21, 2.30-2.43, and 4.13-4.19 ppm, making P(3HB) homopolymer distinct from 3HB. The peak detected near 1.5 ppm is believed to be residual water, and the peak near 7.26 ppm is believed to be deuterated chloroform.

Example 2
<Identification of strain 5-25-4-2>
Species identification: Determination of 16S rRNA base sequence
Nucleic acid was extracted from the 5-25-4-2 strain, precipitated with ethanol, and then the nucleic acid concentration was measured and subjected to agarose gel electrophoresis. DNA extraction was confirmed, and the sample was then subjected to the next step. PCR amplification of 16S rDNA or 18S rDNA was performed on the DNA extract. Because it was not clear whether this strain was a bacterium or a fungus, both 16S rRNA and 18S rRNA primers were used.

PCR was performed using TaKaRa Ex Taq with the following reaction mixture: 0.25 μL of TaKaRa Ex Taq (5 U/μL); 5 μL of 10x Ex Taq Buffer; 4 μL of dNTP mixture (2.5 mM each); 3 μL of primers (10 μM each); 3-5 μL of template (<500 ng); up to 50 μL of sterile MQ water). The reaction conditions were 98°C for 10 seconds, 30 cycles of 55°C for 30 seconds, and 72°C for 90 seconds. A band of approximately 1.5 kbp corresponding to 16S rRNA was confirmed. The PCR product was sequenced to determine a portion of the 16S rDNA sequence. The resulting sequence was then used for a homology search in the BLAST database.

Species Identification: Phenotypic Oxidase Test of Strain 5-25-4-2. A filter paper for cytochrome oxidase testing, "Nissui" (Nissui Pharmaceutical), was placed in a petri dish, and several drops of MQ water were dropped onto the test paper to moisten it completely. The bacterial cells were then applied to the paper with a platinum loop. If the paper turned deep blue within one minute of being left standing, it was judged to be positive; if no color was observed, it was judged to be negative. For the test, a colony of strain 5-25-4-2, grown for 48 hours on PHA synthetic agar medium (Table 14), was used.

Nutritional test: The medium was prepared according to the composition of Ventosa et al. (Table 15) (Anotonio Ventosa, Emilia Quesada, Francisco Rodoriguez-Valera, Francisco Ruiz-Berraquero & Alberto Ramos-cormenzana. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J. Gen. Microbiol. 128, 1959-1968.). The carbon sources used were L-arabinose, D-glucose, glycerol, myo-inositol, D-mannitol, D-mannose, D-sorbitol, and sucrose. The concentration was adjusted to 10% and filter sterilized (Minisart®-RC25, Sartorius, RC, pore size 0.45 μm). All other medium components were sterilized by autoclave. The pH of the medium was adjusted to 7.5 with KOH. The bacterial cells used were stored at 4°C. A colony, including a toothpick, was placed in 5 mL of culture medium and cultured (30°C, shaking at 120 rpm). After that, for those colonies where growth was confirmed, 100 μL of the bacterial solution was added to fresh medium again to determine whether the colony could utilize the carbon source.

Motility test
OF medium was prepared by modifying the composition of Rudolph et al. (Rudolph Hugh, Einar Leifson, "The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria," J. Bacteriol. 1953 Jul;66(1):24-26.) and adjusting the NaCl concentration to 2% (Table 16). The mannitol and glucose used were filter sterilized (Minisart®-RC25, sartorius, RC, pore size 0.45 μm), and sodium alginate and other medium components were autoclaved. The carbon source and medium components were mixed in a clean bench, and the medium was dispensed into test tubes approximately 5 cm in length. The bacterial cells were inoculated using a platinum loop from plate medium stored at 4°C.

Microscopic observations were performed using cultures grown in SW5 liquid medium, PHA synthetic liquid medium, and PHA synthetic agar medium (Table 17). Cobetia sp. strain 5-11-6-3 was used as a control. SW5 liquid medium was prepared according to the method of Antonio et al. with the composition shown in Table 20 (Anotonio Ventosa, Emilia Quesada, Francisco Rodoriguez-Valera, Francisco Ruiz-Berraquero & Alberto Ramos-cormenzana. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J. Gen. Microbiol. 128, 1959-1968.). However, since yeast extract (Difco) was no longer available, yeast extract (Bacto) was used instead. After preparation, 30% marine salts (Table 21) was added to the medium to a final concentration of 5%. The pH was adjusted to 7.5 with KOH and HCl. SW5 liquid medium and PHA synthetic liquid medium samples were inoculated into 10 mL of medium and cultured with shaking at 30°C, 120 rpm, and 48 hours. PHA synthetic agar medium samples were prepared by suspending bacterial cells from a plate stored at 4°C in 100 μL of PHA synthetic liquid medium.

Temperature conditions: Experiments were performed using SW5 medium. The medium composition was prepared according to the method of Antonio et al. (Anotonio Ventosa, Emilia Quesada, Francisco Rodoriguez-Valera, Francisco Ruiz-Berraquero & Alberto Ramos-cormenzana. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J. Gen. Microbiol. 128, 1959-1968). However, since yeast extract (Difco) was no longer available, yeast extract (Bacto) was used instead. After preparation, 30% marine salts was filter-sterilized (Minisart®-RC25, Sartorius, RC, pore size 0.45 μm) and added to the medium to a final concentration of 5%. The pH was adjusted to 7.5 with KOH and NaCl. First, the bacterial cells stored at 4°C were scraped with a toothpick and cultured in SW5 liquid medium (48 hours, 30°C, 120 rpm shaking). The culture medium was then diluted 1/10 with SW5 medium and 100 μL was added. The temperature conditions were 4, 15, 25, 37, 42, and 45°C. For plates grown at 4°C, operations were performed on ice in a clean bench, and the culture was performed in a chamber. For other temperatures, operations were performed at room temperature, and the culture was performed in an incubator.

Growth test was performed on SW5 agar medium to determine the growth concentration of NaCl required for growth. SW medium is hereafter abbreviated as SW-NaCl concentration (%) (e.g., SW5 = SW medium with 5% NaCl). The medium composition was prepared according to the method of Antonio et al. (Anotonio Ventosa, Emilia Quesada, Francisco Rodoriguez-Valera, Francisco Ruiz-Berraquero & Alberto Ramos-cormenzana. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J. Gen. Microbiol. 128, 1959-1968). SW5 liquid medium was prepared with the composition shown in Table 19. SW agar medium with each NaCl concentration was prepared with the composition shown in Table 20. After preparation, 30% marine salts was added to the medium to achieve the desired final concentration. The pH was adjusted to 7.5 with KOH and HCl.
First, bacterial cells stored at 4°C were scraped with a toothpick and cultured in SW5 liquid medium (48 hours, 30°C, 120 rpm shaking). The culture medium was then diluted 1/10 with SW5 medium and 100 μL was added. NaCl conditions were 0, 0.5, 3, 5, 10, 15, 20, and 25%. The culture temperature was 37°C, and growth was determined after one week of culture.

The 1434-bp base sequence of strain 5-25-4-2, a portion of the region flanked by the primers used to amplify the 16S rRNA gene, was determined (Figure 3B). A BLAST homology search was performed. The results showed 99% homology with the 16S rRNA of Cobetia marinana and other species. This suggests that strain 5-25-4-2 may belong to the genus Cobetia (Table 13). Furthermore, the 16S rDNA sequences of strain 5-25-4-2 and Cobetia sp. strain 5-11-6-3 (identified in Patent Application No. 2018-159148) differed by four bases.
The results of examining morphological characteristics and physiological/biochemical properties are summarized in Table 21. When compared with Cobetia marina, the 5-25-4-2 strain was inconsistent with Cobetia marina in the nutritional test in that it was unable to assimilate L-arabinose. However, all morphological characteristics and physiological/biochemical properties were identical to those of Cobetia sp. 5-11-6-3. Therefore, as with the 16S rDNA sequence results, it was concluded that the 5-25-4-2 strain belongs to the genus Cobetia.

Characteristics of Cobetia marina DSM4741 ^T strain (see references), Cobetia sp. 5-1 1-6-3 strain (strain identified in Patent Application No. 2018-159148), and 5-25-4-2 strain Reference: DAVID R. ARAHAL et al., Proposal of Cobetia marina gen. nov., comb. nov., within the Family Halomonadaceae, to Include the Species Halomonas marina System. Appl. Microbiol. 25, 207-211 (2002)
・Lyudmila A. Romanenko, Naoto Tanaka, Vassilii I. Svetashev & Enevold Falsen. (2013). Description of Cobetia amphilecti sp. nov., Cobetia litoralis sp. nov. and Cobetia pacifica sp. nov., classification of Halomonas halodurans as a later heterotypic synonym of Cobetia marina and emended descriptions of the genus Cobetia and Cobetia marina

Example 3
<PHA synthesis experiment using wakame seaweed as raw material>
Experimental Method:
culture
Cobetia sp. 5-11-6-3 or 5-25-4-2 strains were cultured in PHA synthesis medium, and the culture cells were scraped and inoculated into 10 mL of PHA synthesis liquid medium (Table 22). The culture was incubated at 30°C for 2 days with shaking at 120 rpm to prepare a seed culture. Next, 150 mL of PHA synthesis liquid medium (Table 23) containing 2 wt% crushed freeze-dried wakame was prepared in a Sakaguchi flask, and 3 mL of the seed culture was inoculated into each flask. This was incubated at 30°C for 1-2 days with shaking at 120 rpm to prepare a main culture. The crushed freeze-dried wakame added to the medium was placed in an unsealed plastic bag, pre-frozen overnight at -80°C, and then freeze-dried. After freeze-drying, the culture was ground to a powder using a mortar and pestle.
After the main culture, the culture solution was filtered using Kimwipes to separate the seaweed. The bacterial solution was then transferred to a centrifuge tube and centrifuged at 7,700 x g for 15 minutes, after which the supernatant was discarded by decantation. The bacterial cells were then suspended in RO water, centrifuged at 7,700 x g for 10 minutes, and the supernatant was discarded by decantation, a process repeated twice to wash the bacterial cells. The obtained bacterial cells were frozen at -80°C overnight or longer and lyophilized using a freeze dryer (EYELA FDS-1000, Tokyo Rikakikai Co., Ltd.). The experiment was performed in duplicate.

Polymer Extraction: Dried cells were crushed in a mortar and weighed. Approximately 5 mL of chloroform was added per 200-300 mg of dried cells, and extraction was performed at 70°C using a heat block for 48 hours. After extraction, the cells were filtered using a filter (DISMIC®-25HP, Advantec, PTFE, pore size 45 μm) and heated to 70°C using a heat block to dry the extract. If the extract was dirty, it was dissolved in a small amount of chloroform, and an excess amount (approximately 10 times or more) of hexane was added to precipitate the polymer. The hexane was then removed and the extract was dried. The weight of the precipitated polymer was calculated from the weight of the test tube before and after the experiment, and the polymer accumulation rate was calculated from the weight of the dried cells.

^1H -NMR Analysis: 10 mg of the purified polymer was collected on a precision balance to a sample concentration of 5 mg/mL, and added to approximately 2 mL of 0.05 vol% TMS-containing deuterated chloroform (Fujifilm Wako Pure Chemical Industries, Ltd.), then heated to 70°C for dissolution. This analytical sample was placed in a specified amount in an NMR sample tube, and ^1H -NMR analysis was performed using a JNM-AL400 (400 MHz). Tetramethylsilane (TMS) was used as the internal standard, and 20 accumulations were performed.

Cobetia sp. 5-11-6-3 and 5-25-4-2 were cultured in a medium containing 2 wt% pulverized, freeze-dried wakame seaweed. A small amount of a polymer-like white solid was observed in both strains. ^H -NMR analysis of the resulting polymer-like solid confirmed it to be poly-3-hydroxybutanoic acid [P(3HB)], a typical PHA (Figure 4). The P(3HB) accumulation rates were similar, at 4.9 wt% for strain 5-11-6-3 and 4.5 wt% for strain 5-25-4-2 (Table 24). These results demonstrate that strains 5-11-6-3 and 5-25-4-2 can synthesize P(3HB) using dried wakame seaweed as the primary raw material.

Example 4
<PHA synthesis experiment using kelp as a raw material: Examination of the amount of dried kelp added to the culture medium>
Experimental method: Culture
Cobetia sp. strain 5-11-6-3 or Cobetia sp. strain 5-25-4-2 was cultured on agar medium for PHA synthesis, and then the cultured cells were scraped and inoculated into 10 mL of liquid medium for PHA synthesis (Table 22). The culture was then cultured at 30°C for 2 days with shaking at 120 rpm to perform seed culture. Next, 150 mL of liquid medium for PHA synthesis (Table 25) containing 2 or 5 wt% crushed dried kelp was prepared in a Sakaguchi flask, and 3 mL of the seed culture was inoculated into each flask. This was then cultured at 30°C for 1 to 2 days with shaking at 120 rpm to perform main culture. The crushed dried kelp added to the medium was washed with tap water and RO water, then chopped into pieces of approximately 3 x 3 cm. The pieces were placed in an unsealed plastic bag, pre-frozen at -80°C overnight, and then freeze-dried. After freeze-drying, the pieces were crushed at approximately 6,000 rpm using a grinder (New Power MILL PM-2005m, Osaka Chemical Co., Ltd.) to a size of 2.3 mm square.
After the main culture, the culture solution was filtered using Kimwipes to separate the seaweed. The bacterial solution was then transferred to a centrifuge tube and centrifuged at 7,700 x g for 15 minutes, after which the supernatant was discarded by decantation. The bacterial cells were then suspended in RO water, centrifuged at 7,700 x g for 10 minutes, and the supernatant was discarded by decantation, a process repeated twice to wash the bacterial cells. The obtained bacterial cells were frozen at -80°C overnight or longer and lyophilized using a freeze dryer (EYELA FDS-1000, Tokyo Rikakikai Co., Ltd.). The experiment was performed in duplicate.

Polymer Extraction: Dried cells were crushed in a mortar and weighed. Approximately 5 mL of chloroform was added per 200-300 mg of dried cells, and extraction was performed at 70°C using a heat block for 48 hours. After extraction, the cells were filtered using a filter (DISMIC®-25HP, Advantec, PTFE, pore size 45 μm) and heated to 70°C using a heat block to dry the extract. If the extract was dirty, it was dissolved in a small amount of chloroform, and an excess amount (approximately 10 times or more) of hexane was added to precipitate the polymer. The hexane was then removed and the extract was dried. The weight of the precipitated polymer was calculated from the weight of the test tube before and after the experiment, and the polymer accumulation rate was calculated from the weight of the dried cells.

^1H -NMR Analysis: 10 mg of the purified polymer was taken on a precision balance to give a sample concentration of 5 mg/mL, and added to approximately 2 mL of 0.05 vol% TMS-containing deuterated chloroform (Fujifilm Wako Pure Chemical Industries, Ltd.), then heated to 70 °C for dissolution. This analytical sample was placed in a specified amount in an NMR sample tube, and ^1H -NMR analysis was performed using a JNM-AL400 (400 MHz). Tetramethylsilane (TMS) was used as the internal standard, and 20 accumulations were performed.

When Cobetia sp. 5-11-6-3 and Cobetia sp. 5-25-4-2 were cultured in a medium containing 2 wt% of crushed freeze-dried kelp, a polymer-like white solid was observed in both strains. ^H -NMR analysis of the resulting polymer-like solid confirmed that both strains synthesized P(3HB) (Fig. 5).

The P(3HB) accumulation rate was 17.5 wt% for strain 5-11-6-3 and 28.3 wt% for strain 5-25-4-2 (Table 26). Furthermore, when the amount of dried kelp added to the medium was increased from 2 wt% to 5 wt%, the P(3HB) accumulation rate increased to 27.7 wt% for strain 5-11-6-3, but decreased to 5.3 wt% for strain 5-25-4-2. The decrease in P(3HB) accumulation despite the increased amount of dried kelp added to the medium is thought to be due in part to a decrease in the pH of the medium before inoculation when the amount of dried kelp added is increased. Therefore, we next investigated the effect of medium pH on P(3HB) synthesis in medium supplemented with dried kelp.

P(3HB) synthesis by Cobetia sp. strains 5-11-6-3 and 5-25-4-2 in dried kelp-supplemented medium

Example 5
<P(3HB) synthesis experiment using kelp as a raw material: examination of culture medium pH>
Experimental method: Culture
Cobetia sp. 5-11-6-3 or 5-25-4-2 strains were cultured in PHA synthesis medium, and the culture cells were scraped and inoculated into 10 mL of PHA synthesis liquid medium (Table 22). The culture was then cultured at 30°C for 2 days with shaking at 120 rpm to form a seed culture. Next, 150 mL of PHA synthesis liquid medium (Table 25) containing 5 wt% crushed dried kelp was prepared in a Sakaguchi flask, and 3 mL of the seed culture was inoculated into each flask. The initial pH of the medium was set to 5.0, 5.5, or 6.0. After inoculation, the culture was cultured at 30°C for 1 day with shaking at 120 rpm to form a main culture. The initial pH was measured after medium preparation and before autoclaving.

After the main cultivation, the culture medium was filtered using Kimwipes to separate the seaweed. The bacterial liquid was then transferred to a centrifuge tube and centrifuged at 6,500 x g for 15 minutes, after which the supernatant was discarded by decantation. The bacterial cells were then suspended in RO water, centrifuged at 6,500 x g for 10 minutes, and the supernatant was discarded by decantation, a process repeated twice to wash the bacterial cells. The resulting bacterial cells were frozen at -80°C overnight or longer and lyophilized using a freeze dryer (EYELA FDS-1000, Tokyo Rikakikai Co., Ltd.). The experiment was performed in duplicate.

Polymer Extraction: Dried cells were crushed in a mortar and weighed. Approximately 5 mL of chloroform was added per 100 mg of dried cells, and extraction was performed at 70°C using a heat block for 48 hours. After extraction, the cells were filtered using a filter (DISMIC (registered trademark)-25HP, Advantec, PTFE, pore size 45 μm) and heated to 70°C using a heat block to dry the extract. If the extract was dirty, it was dissolved in a small amount of chloroform, and an excess amount (approximately 10 times or more) of hexane was added to precipitate the polymer. The hexane was then removed and the extract was dried. The weight of the precipitated polymer was calculated from the weight of the test tube before and after the experiment, and the polymer accumulation rate was calculated from the weight of the dried cells.

When the initial pH of the medium containing 5 wt% dried kelp was varied, the 5-11-6-3 strain accumulated the highest P(3HB) (48.3 wt%) at pH 5.0 (Table 27). The 5-25-4-2 strain accumulated the highest P(3HB) (50.0 wt%) at pH 6.0. These results demonstrate that the 5-11-6-3 and 5-25-4-2 strains can synthesize P(3HB) using dried kelp as the primary raw material.

This invention is useful in the plastics industry.

SEQ ID NO: 1: 16S rRNA gene nucleotide sequence of microorganism strain 5-25-4-2 SEQ ID NO: 2: 16S rRNA gene nucleotide sequence of microorganism strain 5-11-6-3 SEQ ID NO: 3: 16S rRNA gene nucleotide sequence of microorganism strain 5-28-6-1 SEQ ID NO: 4: 16S rRNA gene amplification primer SEQ ID NO: 5: 16S rRNA gene amplification primer SEQ ID NO: 6: 18S rRNA gene amplification primer SEQ ID NO: 7: 18S rRNA gene amplification primer

Claims (9)

Microorganism deposited under accession number NITE P-03085. A method for producing PHA using seaweed as a raw material, comprising step (a) of culturing a microorganism capable of alginate utilization and polyhydroxyalkanoic acid (PHA) synthesis in a medium containing seaweed, and synthesizing PHA using the seaweed as a carbon source, wherein the microorganism is the microorganism described in claim 1. The manufacturing method described in claim 2, wherein the seaweed is brown algae. The manufacturing method described in claim 3, wherein the brown algae belong to the Laminariales or Fucales order. The production method according to any one of claims 2 to 4, wherein the culturing step (a) is carried out in a medium having a pH in the range of 3.0 to 9.0 before the start of culturing. The method according to any one of claims 2 to 5 , further comprising a step (b) of extracting PHA from the culture obtained in the culture step (a). The method according to any one of claims 2 to 6 , wherein the PHA comprises a 3-hydroxybutanoic acid monomer unit. The method according to claim 7 , wherein the PHA is poly(3-hydroxybutanoic acid). A method for producing a bioplastic product, comprising the steps of: carrying out the method according to any one of claims 2 to 8 to obtain polyhydroxyalkanoic acid (PHA); and obtaining a plastic product containing the PHA obtained in the step.